Treatment of Neurodegenerative Diseases Through Inhibition of HSP90

ABSTRACT

Treatment of neurodegenerative diseases is achieved using small molecule purine scaffold compounds that inhibit Hsp90 and that possess the ability to cross the blood-brain barrier or are other wise delivered to the brain.

CLAIM FOR PRIORITY

This application claims the priority benefit of U.S. ProvisionalApplication No. 60/806,427, filed Jun. 30, 2006, which is incorporatedherein by reference for all purposes.

STATEMENT OF FEDERAL FUNDING

This invention was supported in part by NIH grant AG09464. The UnitedStates government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

This application relates to the treatment of neurodegenerative diseasesthrough inhibition of heat shock protein 90 (HSP90).

The HSP90 family of proteins has four recognized members in mammaliancells: Hsp90α and β. Grp94 and Trap-1. Hsp90α and β exist in the cytosoland the nucleus in association with a number of other proteins. Hsp90 inits various forms is the most abundant cellular chaperone, and has beenshown in experimental systems to be required for ATP-dependent refoldingof denatured or “unfolded” proteins. It has therefore been proposed tofunction as part of the cellular defense against stress. When cells areexposed to heat or other environmental stresses, the aggregation ofunfolded proteins is prevented by pathways that catalyze their refoldingor degradation. This process depends on the association of the unfoldedprotein in an ordered fashion with multiple chaperones (Hsp 60, 90 and70 and p23), forming a “refoldosome” and ultimately the ATP-dependentrelease of the chaperones from the refolded protein.

Hsp90 may also play a role in maintaining the stability and function ofmutated proteins. It seems to be required for expression of mutated p53and v-src to a much greater extent than for their wild-typecounterparts. It has been suggested that this occurs as a result ofHsp90-mediated suppression of the phenotypes of mutations that lead toprotein unfolding.

Hsp90 is also necessary to the conformational maturation of several keyproteins involved in the growth response of the cell to extracellularfactors. These include the steroid receptors as well as certaintransmembrane kinases (i.e., Raf serine kinase, v-src and Her2). Themechanism whereby Hsp90 affects these proteins is not fully understood,but appeal's to be similar to its role in protein refolding. In the caseof the progesterone receptor, it has been shown that binding and releaseof Hsp90 from the receptor occurs in a cyclic fashion in concert withrelease of other chaperones and immunophilins and is required for highaffinity binding of the steroid to the receptor. Thus, Hsp90 couldfunction as a physiologic regulator of signaling pathways, even in theabsence of stress.

Hsp90 has been shown to be overexpressed in multiple tumor types and asa function of oncogenic transformation. Whether it plays a necessaryrole in maintaining transformation is unknown, but it could have atleast three functions in this regard. Cancer cells grow in anenvironment of hypoxia, low pH and low nutrient concentration. They alsorapidly adapt to or are selected to become resistant to radiation andcytotoxic chemotherapeutic agents. Thus, the general role of Hsp90 inmaintaining the stability of proteins under stress may be necessary forcell viability under these conditions. Secondly, cancer cells harbormutated oncogenic proteins. Some of these are gain-of-function mutationswhich are necessary for the transformed phenotype. Hsp90 may be requiredfor maintaining the folded, functionally-active conformation of theseproteins. Thirdly, activation of signaling pathways mediated by steroidreceptors, Raf and other Hsp90 targets is necessary for the growth andsurvival of many tumors which thus probably also require functionalHsp90.

Neurodegeneration, similar to cancer, is likely not the result of asingle dysregulatory event, but rather a several-step process involvingenvironmental, epigenetic and genetic events that lead to creation of acomplex transformed phenotype manifested by abnormal expression,post-translational modification and processing of certain proteins. Thefunctional maintenance of these dysregulated proteins in neurons mayrequire, analogously to the cancer afflicted cell, the regulatorymechanism of molecular chaperones to evolve along with the transformingprocess.

In the context of neurodegenerative diseases, Hsp90 may play two roles.First, aberrantly activated kinases (such as cdk5/p35, gsk3beta) inneurodegenerative diseases may require Hsp90 for functioning. Thus,Hsp90 inhibition may restore damaged signaling networks in the diseasedbrain by alleviating aberrant phosphorylation, leading to reducedaberrant protein aggregation, and elimination or reduction of aggregatesand of their associated toxicity. Second, pathogenic mutants (such as ofAPP or presenilins in AD or mtau in FTDP-17 or mutant androgen receptorin bulbar muscular atrophy) may require Hsp90 for correct folding andfunctioning, thus Hsp90 inhibition may lead to the elimination of theseproteins and result in reduction of aggregates and consequent plaque ortangle formation.

Most neurodegenerative diseases are probably characterized by bothmutants and aberrant signaling, and Hsp90 can play a role with respectto pathogenic mutants as well. Tau mutations cause autosomal dominantfrontal temporal dementia. Pathologies linked to mutations of theandrogen receptor include the complete androgen insensitivity syndrome(CAIS) and the spinal and bulbar muscular atrophy (SBMA or Kennedy'sdisease). (4) Mutations in the presenilin genes are the major cause offamilial AD. Analysis of conditional knockout mice has shown thatinactivation of presenilins results in progressive memory impairment andage-dependent neurodegeneration, suggesting that reduced presenilinactivity might represent an important pathogenic mechanism. Presenilinspositively regulate the transcription of cAMP response element(CRE)-containing genes, some of which are known to be important formemory formation and neuronal survival. (5) Alzheimer's Disease (AD) ischaracterized both by NFTs (tau aggregates) and plaques (AP deposits).In Alzheimer's disease, mutations in amyloid precursor protein or in thepresenilins cause autosomal dominant disease. These are the substrateand proteases responsible for the production of the deposited peptideAβ. Prion mutations cause Gerstmann Straussler syndrome and hereditaryCreutzfeldt-Jakob disease, alpha-synuclein mutations cause autosomaldominant Parkinson's disease. In these cases, the pathogenic mutation isin the protein that is deposited in the diseased tissue and the wholeprotein is deposited. Huntington D results from a mutant huntingtin. (9)Thus, in all the cases, the mutations lead to the disease by a mechanismthat involves the deposition process.

These characteristics of Hsp90 make it a viable target for therapeuticagents. HSP90 family members possess a unique pocket in their N-terminalregion that is specific to and conserved among all Hsp90s from bacteriato mammals, but which is not present in other molecular chaperones. Theendogenous ligand for this pocket is not known, but it binds ATP and ADPwith low affinity and has weak ATPase activity. The ansamycinantibiotics geldanamycin (GM) and herbimycin (HA) have been shown tobind to this conserved pocket, and this binding affinity has been shownfor all members of the HSP90 family. International Patent PublicationNo. WO98/51702 discloses the use of ansamycin antibiotics coupled to atargeting moiety to provide targeted delivery of the ansamycin leadingto the degradation of proteins in and death of the targeted cells.International Patent Publication No. WO00/61578 relates to bifunctionalmolecules having two moieties which interact with the chaperone proteinHsp90, including in particular homo- and heterodimers of ansamycinantibiotics. These bifunctional molecules act to promote degradationand/or inhibition of HER-family tyrosine kinases and are effective fortreatment of cancers which overexpress Hcr-kinases.

Exemplary small molecule therapeutics that bind to the same bindingpocket of Hsp90 as ATP and the ansamycin antibiotics are disclosed inPCT Publication No. WO02/36075, PCT Application No. PCT/US06/03676 andUS Patent Publications 2005-0113339, 2005-0004026, 2005-0049263,2005-0256183, 2005-0119292, 2005-0113340 and 2005-0107343, all of whichare incorporated herein by reference.

In aged organisms, chaperone overload leads to a significant decrease inthe robustness of cellular networks shifting their function towards amore stochastic behavior. Unbalanced chaperone requirement and chaperonecapacity helps the accumulation of misfolded and aggregated proteinsespecially in the nervous system, due to the very limited proliferationpotential of neurons. In addition, damaged signaling networks lose theiroriginal stringency, and irregular protein phosphorylation occurs. Anappealing approach to alleviating and reversing such damaging effects isby modulating Hsp90 activity. Inhibitors of Hsp90 activity release HSF1from Hsp90 complexes and correct the defective regulation of HSF1activity after heat stress leading to an increase in cellular levels ofchaperones, such as Hsp70 and Hsp40. Overexpression of these chaperoneshas been shown to represent a general way of reinstating proper foldingand alleviating misfolded proteins' toxic effects. In addition to theireffects on reinstating correct folding, Hsp90 inhibitors may regulateproteins involved in signaling networks of diseased neurons.

The usefulness of Hsp90 inhibitors as clinical agents in the treatmentof neurodegenerative diseases, however, will depend on whether theireffects occur at concentrations of drug that are tolerable to thepatient and on whether the drugs can be administered in such a fashionso as to achieve these concentrations in the brain. Unfortunately, knownHsp90 inhibitors such as geldanamycin and 17AAG, its derivative in PhaseI clinical trial for cancer, and the unrelated compound radicicol havesignificant limitations. They are poorly soluble, difficult to formulateand do not cross the blood-brain barrier. Thus, in order to realize thepotential for treatment of neurodegenerative diseases, therapeuticagents that inhibit Hsp90, and that have sufficient solubility and theability to cross the blood-brain barrier or otherwise be delivered tothe brain are needed.

SUMMARY OF THE INVENTION

In accordance with the present invention, treatment of neurodegenerativediseases is achieved using small molecule purine scaffold compounds thatinhibit Hsp90 and that possess the ability to cross the blood-brainbarrier. Thus, in accordance with the present invention, there isprovided a method for treatment of neurodegenerative disease comprisingthe step or administering to an individual in need of such treatment aneffective amount of a purine-scaffold compound that inhibits Hsp90, andthat crosses the blood-brain barrier or is otherwise delivered to thebrain.

In one embodiment, the purine scaffold compound used in the method ofthe invention has a purine moiety connected at the 8- or 9-position viaa linker to a monocyclic substituent group. Such compounds are describedin PCT Publication No. WO02/36075, PCT Application No. PCT/US06/03676and US Patent Publications 2005-0113339, 2005-0004026, 2005-0049263,2005-0256183, 2005-0119292, 2005-0113340 and 2005-0107343.

In one embodiment, the method of the invention makes use of a smallmolecule purine scaffold compound has the general structure:

wherein R is hydrogen, a C₁ to C₁₀ alkyl, alkenyl, alkynyl, or analkoxyalkyl group, optionally including heteroatoms such as N or O,optionally connected to the 2′-position to form an 8 to 10 member ring;Y₁ and Y₂ are independently C, N, S or O, with the proviso that when Y₁and/or Y₂ is O the double bonds are missing or rearranged to retain thearyl nature of the ringX₄ is hydrogen, halogen, for example F or Cl, or Br;X₃ is CH₂, CF₂, S, SO, SO₂, O, NH, or NR², wherein R² is alkyl; andX₂ is halogen, alkyl, halogenated alkyl, alkoxy, halogenated alkoxy,hydroxyalkyl, pyrollyl, optionally substituted aryloxy, alkylamino,dialkylamino, carbamyl, amido, alkylamido dialkylamido, acylamino,alkylsulfonylamido, trihalomethoxy, trihalocarbon, thioalkyl, SO₂,alkyl, COO-alkyl, NH₂, OH, or CN or part of a ring formed by R; andX₁ represents one more substituents on the aryl group, with the provisothat X₁ represents at least one substituent in the 5′-position saidsubstituent in the 5′-position being selected from the same choices asX₂: C₁ to C₆ alkyl or alkoxy; or wherein X₁ has the formula—O—(CH₂)_(n)—O—, wherein n is 1 or 2, and one of the oxygens is bondedat the 5′-position of the aryl ring and the other is bonded to the 4′position.

The ride-side aryl group may be phenyl, or may include one or moreheteroatoms. For example, the right-side aryl group may be anitrogen-containing aromatic heterocycle such as pyrimidine.

In specific embodiments of the composition of the invention, theright-side aryl group is substituted at the 2′ and 5′ position only. Inother embodiment, the right side aryl group is substituted at the 2′,4′, and 5′ positions. In yet other embodiments, the right side arylgroup is substituted at the 4′ and 5′ positions only. As will beappreciated by persons skilled in the art, the numbering is based on thestructure as drawn, and variations in the structure such as theinsertion of a heteroatom may alter the numbering for purposes of formalnomenclature.

In other specific embodiments of the composition of the invention, theright side aryl group has a substituent at the 2′-position and X₁ hasthe formula —X—Y—Z— with X and Z connected at the 4′ and 5′ positions tothe right side aryl, wherein X, Y and Z are independently C, N, S or O,connected by single or double bonds and with appropriate hydrogen, alkylor other substitution to satisfy valence. In some embodiments, at leastone of X, Y and Z is a carbon atom. In one specific embodiment. X₁ is—O—(CH₂)_(n)—O—, wherein n is 1 or 2, and one of the oxygen atoms isbonded at the 5′-position of the aryl ring and the other at the 4′position. Additional examples of compounds of this type are shown in theFIG. 4.

In accordance with specific embodiments of the invention, the purinescaffold composition has a formula as shown in FIG. 5.

The composition of the invention may also be a homodimer or heterodimerof these compounds having the formula:

provided that the compound retains the ability to inhibit hsp90 and alsoto cross the blood brain barrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows tau phosphorylation activity in mouse brain followingshort term administration of PU24FCl.

FIG. 1B shows concentration of PU24FCl in mouse brain following shortterm administration.

FIG. 2 shows the effects of long-term Hsp90 inhibition with PU24FCl ontau phosphorylation and expression of other proteins.

FIG. 3 shows the effects of long-term Hsp90 inhibition with PU-DZ8 ontau phosphorylation.

FIG. 4 shows compounds useful in the method of the invention.

FIG. 5 shows compounds useful in the method of the invention.

FIG. 6 shows a synthetic scheme for making compounds useful in theinvention.

FIG. 7 shows a synthetic scheme for making compounds useful in theinvention.

FIGS. 8A and B shows levels of various proteins in the brains of micetreated in accordance with the invention by intraperitonealadministration of a purine scaffold compound.

FIG. 9 shows degradation of the mutant protein, mtau (HT7) after onedose administration of PU-DZ8. It also shows the change in chaperonelevels (hsp70 increase) and kinase expression (p35 levels).

FIG. 10 shows the dependency of mutant tau protein on hsp90 chaperoning.

FIGS. 11 A and B show hsp90 binding and hsp70 induction by purinescaffold compounds in neuroblastoma cells.

FIG. 12 shows the binding affinity of PU-DZ8, PU24FCl and 17AAG to hsp90in JNPL3 brain extracts.

FIG. 13 shows that PU-DZ8 reaches pharmacologically relevantconcentrations in JNPL3 transgenic mouse brain following administrationof one dose of 75 mg/kg PU-DZ8 administered i.p.

FIG. 14A shows the effects of one dose, short term administration ofPU-DZ8 on the levels of soluble mutant tau in the JNPL3 mouse brain Thesubcortical brain region of 2.5 to 4-month old mice is presented. HumanTau levels were normalized to those of Hsp90.

FIG. 14B shows the effect of one dose, short-term administration ofPU-DZ8 on the levels of insoluble mutant tau in the JNPL3 mouse brain.Analysis of the insoluble tau (P3) fractions extracted from thesubcortical brain region of 6-month old mice treated with PU-DZ8 (75mg/kg) for 4, 8, 12 and 24 h is presented.

FIG. 15 shows the effect of long term PU-DZ8 administration onhyperphosphorylated tau in toxic tau aggregates.

FIG. 16A shows the effect of PU-DZ8 on p35 in the htau mice that expresspathogenically hyperphosphorylated WT tau similarly to Alzheimer'spatients

FIG. 16B shows the effect of PU-DZ8 tau phosphorylation in the htau micethat express pathogenically hyperphosphorylated WT tau similarly toAlzheimer's patients

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for treatment ofneurodegenerative disease, comprising the step of administering to anindividual in need of such treatment a therapeutically effective amountof a purine scaffold compound that inhibits Hsp90 and that crosses theblood-brain barrier or is otherwise delivered to the brain.

As used in this application, the term “treatment” refers to delaying theonset of symptoms, reducing the severity or delaying the symptomaticprogression of neurodegenerative disease in the individual. A cure ofthe disease is not required to fall within the scope of treatment.Further, it will be appreciated that the specific results of thesetreatment goals will vary from individual to individual, and that someindividuals may obtain greater or lesser benefits than the statisticalaverage for a representative population. Thus, treatment refers toadministration of composition to an individual in need, with theexpectation that they will obtain a therapeutic benefit.

The term “neurodegenerative disease” refers to disease characterized byabnormalities in signaling pathways, for example aberrantphosphorylation due to dysregulated kinase activity, mutant proteins(mutant tau, mutant APP) and chaperone unbalance leading to misfoldingand increased apoptosis. In a specific embodiment, the neurodegenerativedisease is a tauopathy, i.e. neurodegenerative disease characterized bytau protein abnormalities that share the feature of hyperphosphorylatedtau protein, and intracellular neurofibrillary tangle (NFT) formation.Without limitation, the term “neurodegenerative disease” as used in thisapplication refers to and encompasses Alcohol-induced neurodegeneration(10)); Alzheimer's disease (11); Amyotrophic lateral sclerosis (13; 14);Brain ischemia (15; 20); Cocaine addiction (21); Diffuse Lewy bodydisease (22); Electroconvulsive seizures (23); Fetal alcohol syndrome(10); Focal cortical dysplasia (24); Hereditary canine spinal muscularatrophy (25); Inclusion body myositis (26); Multiple system atrophy (27;28); Niemann-Pick type C; Parkinson's disease (22); and Peripheral nerveinjury (71).

The term “administering” refers to the act of introducing into theindividual the therapeutic compound. In general, any route ofadministration can be used. Because the compounds used in the method ofthe invention may be capable of crossing the blood-brain barrier,systemic administration can be used. Thus, in certain embodiment of theinvention, administration by oral, intravenous, intramuscular orparenteral injection is appropriate. Administration may also be done tothe brain by inhalation because there is a compartment at the upper sideof the nose that connects with the brain without having the BBBcapillaries. Compounds that cross the blood brain barrier are preferredfor this mode of administration as well, although this characteristicsis not strictly required.

The term “therapeutically effective amount” encompasses both the amountof the compound administered and the schedule of administration that ona statistical basis obtains the result of preventing, reducing theseverity or delaying the progression of neurodegenerative disease in theindividual. As will be appreciated, preferred amounts will vary fromcompound to compound in order to balance toxicity/tolerance withtherapeutic efficacy and the mode of administration. Determination ofmaximum tolerated dose and of the treatment regime in terms of numberand frequency of dosing is a routine part of early clinical evaluationof a compound.

The term “crosses the blood brain barrier” as used herein refers to theability of the compound to transit to the brain in detectable amountsfollowing systemic administration. The ability of a compound to crossthe blood brain barrier can be assessed using animal models such as miceAs illustrated in the examples below, a single dose administration, forexample at 50 to 200 mg/kg, can be employed, with animals sacrificed atintervals and the brain concentration of the compound determined. Itwill be appreciated that the extent to which a compound does transit tothe brain will also have an impact on the amount of the therapeuticcompound that is necessary. In general, however, compounds that crossthe blood brain barrier will have molecular weights of less than 400daltons, a degree of lipid solubility, preferably comparable to thecompounds disclosed herein, the absence of restrictive plasma proteinbindings and the absence of significant affinity for any of the severalBBB active efflux transporters such as p-glycoprotein. In this regard,it is noted that 17-AAG does not effectively cross the blood brainbarrier and is a P-glycoprotein substrate.

The therapeutic compound employed in the method of the present inventionis suitably a small molecule purine scaffold compounds that inhibitHsp90 and that possess the ability to cross the blood-brain barrier. Theterm “purine scaffold compound” refers to a compound that has a purinemoiety that to which is bonded an additional aryl or heteroaryl ring atthe 8- or 9-position, wherein the compound as a whole possesses thenecessary flexibility and substituent groups to be received within theN-terminal pocket of Hsp90. These general requirements are discussed inPCT Publication No. WO02/36075.

In one embodiment, the method of the invention makes use of a smallmolecule purine scaffold compound has the general structure:

wherein R is hydrogen, a C₁ to C₁₀ alkyl, alkenyl, alkynyl, or analkoxyalkyl group, optionally including heteroatoms such as N or O,optionally connected to the 2′-position to form an 8 to 10 member ring,Y₁ and Y₂ are independently C, N, S or O, with the proviso that when Y₁and/or Y₂ is O the double bonds are missing or rearranged to retain thearyl nature of the ringX₄ is hydrogen, halogen, for example F or Cl, or Br;X₃ is CH₂, CF₂, S, SO, SO₂, O, NH, or NR², wherein R² is alkyl; andX₂ is halogen, alkyl, halogenated alkyl, alkoxy, halogenated alkoxy,hydroxyalkyl, pyrollyl, optionally substituted aryloxy, alkylamino,dialylamino, carbamyl, amido, alkylamido dialkylamido, acylamino,alkylsulfonylamido, trihalomethoxy, trihalocarbon, thioalkyl, SO₂,alkyl, COO-alkyl, NH₂, OH, or CN or part of a ring formed by R; andX₁ represents one more substituents on the aryl group, with the provisothat X₁ represents at least one substituent in the 5′-position saidsubstituent in the 5′-position being selected from the same choices asX₂: C₁ to C₆ alkyl or alkoxy; or wherein X₁ has the formula—O—(CH₂)_(n)—O—, wherein n is 1 or 2, and one of the oxygens is bondedat the 5′-position of the aryl ring and the other is bonded to the 4′position.

The ride-side aryl group may be phenyl, or may include one or moreheteroatoms. For example, the right-side aryl group may be anitrogen-containing aromatic heterocycle such as pyrimidine.

In specific embodiments of the composition of the invention, theright-side aryl group is substituted at the 2′ and 5′ position only. Inother embodiment, the right side aryl group is substituted at the 2′,4′, and 5′ positions. In yet other embodiments, the right side arylgroup is substituted at the 4′ and 5′ positions only. As will beappreciated by persons skilled in the art, the numbering is based on thestructure as drawn, and variations in the structure such as theinsertion of a heteroatom may alter the numbering for purposes of formalnomenclature.

In other specific embodiments of the composition of the invention, theright side aryl group has a substituent at the 2′-position and X₁ hasthe formula —X—Y—Z— with X and Z connected at the 4′ and 5′ positions tothe right side aryl, wherein X, Y and Z are independently C, N, S or O,connected by single or double bonds and with appropriate hydrogen, alkylor other substitution to satisfy valence. In some embodiments, at leastone of X, Y and Z is a carbon atom. Y in —X—Y—Z may also be —(CH₂)₂ suchthat the X—Y—Z group forms a six-membered ring. In one specificembodiment, X₁ is —O—(CH₂)_(n)—O—, wherein n is 1 or 2 from 0 to 2, andone of the oxygen atoms is bonded at the 5′-position of the aryl ringand the other at the 4′ position. Additional examples of compounds ofthis type are shown in the FIG. 4.

In specific embodiments of the invention, R is 3-isopropylaminopropyl,

-   3-(isopropyl(methyl)amino)propyl, 3-(isopropyl(ethyl)amino)propyl,-   3-((2-hydroxyethyl)(isopropyl)amino)propyl,    3-(methyl(prop-2-ynyl)amino)propyl.-   3-(allyl(methyl)amino)propyl, 3-(ethyl(methyl)amino)propyl.-   3-(cyclopropyl(propyl)amino)propyl,    3-(cyclohexyl(2-hydroxyethyl)amino)propyl,-   3-(2-methylaziridin-1-yl)propyl, 3-(piperidin-1-yl)propyl,-   3-(4-(2-hydroxyethyl)piperazin-1-yl)propyl, 3-morpholinopropyl,-   3-(trimethylammonio)propyl, 2-(isopropylamino)ethyl,    2-(isobutylamino)ethyl,-   2-(neopentylamino)ethyl, 2-(cyclopropylmethylamino)ethyl,    2-(ethyl(methyl)amino)ethyl,-   2-(isobutyl(methyl)amino)ethyl, or    2-(methyl(prop-2-ynyl)amino)ethyl.

In accordance with specific embodiments of the invention, the purinescaffold composition has a formula as shown in FIG. 5.

The composition of the invention may also be a homodimer or heterodimerof these compounds having the formula:

provided that the compound retains the ability to inhibit hsp90 and alsoto cross the blood brain barrier.

Where the active compound in vivo is the dimeric form, the compoundretains the ability to inhibit hsp90 and also to cross the blood brainbarrier. In this case, the linker may be any generally linear group ofatoms that provides the two parts of the dimer with sufficient rotationfreedom to allow both to interact independently with an N-terminalpocket of HSP90. Non-limiting examples of suitable linkers include C₄ toC₁₀ alkyl, alkenyl or alkynyl groups, and secondary amines having atotal length of 4 to 10 atoms.

Compounds of this type may also be provided with a degradable orcleavable linker, such that monomeric agents are provided in vivo. Inthis embodiment, the dimeric form need not retain activity or theability to cross the blood brain barrier, and the nature of the linkertherefore is not relevant to activity, only to the ability to formactive monomeric species. In general, moderately lipophilic drugs suchas PUs cross the BBB by passive diffusion. A good structuralunderstanding for the BBB permeability is still lacking, but severalparameters are believed to facilitate such behavior. Lipophilicity wasthe first of the descriptors to be identified as important for CNSpenetration. For several classes of CNS active substances, Hansch andLeo (89) found that blood-brain barrier penetration is optimal when theLogP values are in the range of 1.5-2.7, with the mean value of 2.1. Themean value for ClogP for the marketed CNS drugs is 2.5. PU-DZ8 has acalculated logP value of 1.73 (using Molinspiration) and anexperimentally determined value of 1.53 (using RP-HPLC). CNS drugs havesignificantly reduced molecular weights (MW) compared with othertherapeutics. The rules for molecular weight in CNS drugs have beenreviewed, where small molecules may undergo significant passivelipid-mediated transport through the blood brain barrier, when themolecular mass is kept in or below a 400- to 600-Da range (90). PU-DZ8has a MW of 512. All the QSAR equations emphasize the importance ofhydrogen bonding—CNS penetration requires 5 or less hydrogen bondacceptors (91). PU-DZ8 has 4. PSA has been shown to be a very gooddescriptor characterizing drug absorption, including intestinalabsorption, bioavailabhility, Caco-2 permeability and BBB penetration.PSA has been used as a predictor for BBB penetration by manyinvestigators (92). In general, drugs aimed at the CNS tend to havelower polar surface areas than other classes (93.94). PSA for CNS drugsis significantly less than for other therapeutics with PSA for CNSpenetration estimated at 60-70 Å² through 90 Å² (95.96). The upper limitfor PSA for a molecule to penetrate the brain is around 90 Å². DZ8 has aPSA of 104 Å². Changing the nature of the chain attached to the 9Nposition from a secondary to a tertiary amine drops the PSA to 90 Å².Number of rotatable bonds has been shown to be a very good descriptor oforal bioavailability of drugs (97-99). It is suggested that compoundswhich meet only the two criteria of (1) 10 or fewer rotatable bonds and(2) polar surface area equal to or less than 140 Å² (or 12 or fewerH-bond donors and acceptors) will have a high probability of good oralbioavailability in the rat (99). Many CNS drugs are basic and exist inequilibrium between their charged and neutral states under physiologicalconditions or are amphiphilic if they also possess an acidic group.Possession of a positive charge at pH 7-8 tends to favor brainpermeation (100). Additionally, compounds possessing a tertiary nitrogen(a feature of many CNS drugs) show a higher degree of brain permeation.All these characteristics are modeled into purine scaffold compounds asdescribed herein.

Another characteristic which is indicative of the ability to cross theblood brain barrier is protein binding. Drug-protein interaction is areversible process and a successful CNS drug should not be an efficientP-glycoprotein substrate (in vivo) (102). It is not sufficient for apotential neurotherapeutic agent to move across the BBB—it also has tostay in the brain long enough to exert its desired action. This meansthat it also has to avoid being a substrate for a variety of transportproteins that work to extrude compounds from the brain. The Hsp90inhibitor 17AAG is a P-gp substrate, however the purine scaffoldtherapeutic PU-DZ8 is not a substrate of P-pg and thus is not readilyextruded from the brain by this mechanism.

Synthetic methods for making compounds useful in the method of theinvention are described in PCT Publication No. WO02/36075. PCTApplication No. PCT/US06/03676 and US Patent Publications 2005-0113339,2005-0004026, 2005-0049263, 2005-0256183, 2005-0119292, 2005-0113340 and2005-0107343. FIGS. 6 and 7 shows synthetic schemes for making compoundswith the structures as shown in FIG. 4. In the case of a carbon linker,phenylacetic acids are first generated by replacing the methylenedioxybridge with the metabolically stable isosters depicted in FIG. 6.Synthesis commences by coupling 2,4,5,6-tetraaminopyrimidine with theacid fluoride of the corresponding carboxylic acid. The acid fluoride isgenerated by treating the phenylacetic acid with cyanuric fluoride andpyridine in CH₂Cl₂. Following a quick water wash, the resulted acidfluoride is used in the next step without further purification. Theamide resulted from the pyrimidine-acid fluoride coupling is cyclized toby heating in alcoholic NaOMc. Transformation of the C2-amino group tofluorine (NH₂ to F) is conducted by a modified Schiemanndiazotization-fluorodediazoniation of the amino derivative inHF/pyridine in the presence of NaNO₂. We and others have previouslydetermined that fluorine in this position in general augmented thepotency of the resulting purines, likely by increasing the hydrogendonor ability of C6 NH2. Further selective halogenation using either NISor NBS leads to the corresponding iodo- or bromo-derivatives. These arealkylated first with 1,3-dibromopropane or 1,2-dibromobutane in thepresence of Cs₂CO₃. Formation of dimer is not detected in this reaction.The resulted bromine is further alkylated in the presence of excessR₁R₂NH to give the final product.

For derivatives containing a sulfur linker, synthesis is carried outusing a method previously described by He et al (1) and employs thecopper catalyzed coupling of 8-mercaptoadenine with the aryliodide (FIG.7). The reaction occurs in anhydrous DMF at 110° C. under nitrogen. The8-arylsulfanyl adenine is further iodinated selectively at position 2 ofthe aryl moiety using NIS as a source of electrophylic iodine and TFA asa catalyst. This is further alkylated at 9N in the presence of excessR₁R₂NH to give the final product.

Application of the Invention to Tauopathies

Alzheimer's disease (AD) is the most common neurodegenerative disordercharacterized by the progressive deterioration of cognition and memoryin association with the presence of senile plaques, neurofibrillarytangles, and massive loss of neurons, primarily in the cerebral cortexand hippocampus. Senile plaques are extracellular deposits composed ofβ-amyloid (Aβ) fibrils, surrounded by dystrophic neurites, reactivemicroglia and astrocytes. Filamentous Tau inclusions are increasinglyrecognized as the hallmark of tauopathies, a growing family ofneurodegenerative diseases including AD, Down's syndrome (DS), severalvariants of prion diseases, progressive supranuclear palsy (PSP),amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam(ALS/PDC), sporadic frontotemporal dementia with parkinsonism (FTDP),Pick's disease and familial FTDP-17 syndromes. Tau is a criticalcomponent of the neuronal cytoskeleton. Some of the morphologicalchanges associated with neuronal apoptosis involve a significantmodification of the cytoskeletal network, likely to contribute to thesubsequent degeneration of neurons, indicating disruption ofcytoskeletal network can cause neurodegeneration. In axons, tau proteinis one of the predominant microtubule associated proteins (30). Itstabilizes microtubules and promotes neurite outgrowth. This apparentlybeneficial role of tau contrasts with its anomalous behavior in severalneurodegenerative diseases, most prominently AD, where it occurs in ahighly phosphorylated form, detaches from microtubules, and aggregates.Pathogenic tau mutations or abnormal tau phosphorylation (which occursin AD and frontotemporal dementias) result in a more rapid developmentof NFTs and neurologic disease, a feature consistent with the view thatthese diseases result from tau aggregation (31).

Several mutations in human tau isoforms on chromosome 17 result in acluster of neurodegenerative diseases, termed “frontotemporal dementiaand parkinsonism linked to chromosome 17 (FTDP-17)” and arecharacterized by the accumulation of neurofibrillary tangles similar tothose in AD, in affected brain regions. Biochemical studies of these taumutants reveal that they are less stable than normal tau and tend toform fibrillar aggregates (32), consistent with the view thattauopathies are diseases related to protein folding and stability. Thetau proteins in AD are not mutated, yet nevertheless comprise NFTs. InAD, tau becomes hyperphosphorylated, and it has been hypothesized thatthis impairs the microtubule stabilizing role of tau

Hyperphosphorylated tau is believed to misfold, undergo net dissociationfrom microtubules, form abnormal filamentous aggregates (paired helicalfilaments, PIIFs) and polymerize into NFTs (33). The central role ofprotein misfolding in this process is illustrated by observations thatthe different tau mutations linked to FDTP-17 differ in their levels ofphosphorylation and in their effects on microtubules (34). We have shownan inverse relationship between aggregated tau and the levels of heatshock protein (Hsp)70/90 in tau transgenic mice and Alzheimer's diseasebrains. In various cellular models, increased levels of Hsp70 and Hsp90promoted tau solubility and tau binding to microtubules, reducedinsoluble tau and caused reduced tau phosphorylation. Conversely,lowered levels of Hsp70 and Hsp90 resulted in the opposite effects. Wehave also demonstrated a direct association of the chaperones with tauproteins. Our results suggested that up-regulation of molecularchaperones may suppress formation of neurofibrillary tangles bypartitioning tau into a productive folding pathway and therebypreventing tau aggregation (12).

Hsp90 inhibitors were found to beneficially increase levels of Hsp70chaperone in other neurodegenerative systems. Induction of chaperones,especially Hsp70 and Hsp40, was found to delay the onset or to diminishthe symptoms in folding diseases (3). GM was found to activate a heatshock response and inhibit huntingtin aggregation in a cell culturemodel of Huntington's disease (16). GM was reported to restore functionto a defective heat shock response in scrapie-infected cells (17, 18).Auluck et al (19) reported that treatment of a fly model of Parkinson'sdisease with GM fully protected against α-synuclein toxicity. Theseeffects were seen without altering the microscopic appearance ofneuronal inclusions, suggesting that chaperones “detoxify” the proteinsaggregates in a more subtle way than just preventing the formation ofprotein aggregates. Auluck also suggested that only a modest change orredistribution of chaperones might be sufficient for neuroprotection(19).

These effects of the Hsp90 inhibitors occur by their modulation of theHSF1-hsp9) complexes. In normal cells, the presence of misfolded oraggregated proteins triggers a complex biological response, referred toas the heat shock response (6) This involves the expression of heatshock proteins (HSPs, molecular chaperones) and of proteins involved inthe ubiquitin-proteasome pathway. The evolution of such complexmachinery testifies to the fact that is necessary for cells to isolateand rapidly clear unfolded proteins as soon as they appear. Inunstressed cells, HSF1 forms a dynamic complex with Hsp90 (7). Whenprotein unfolding increases, these non-native proteins compete with HSF1for Hsp90 binding resulting in an increase in unbound HSF1 and inductionof HSPs. When stress-induced synthesis of chaperones is impaired foldingdiseases are possible (8). As suggested by its regulation of HSF1activity, interference with Hsp90 activity by Hsp90 inhibitors triggersa heat shock response. The activity of neuronal disease-activatedkinases is regulated by Hsp90.

We have also shown that tau phosphorylation levels at pathological siteswas reduced after treatment with the Hsp90 inhibitor geldanamycin (GM)in AD cellular models. Cdk5, Gsk3 and MAPK are three major kinases thatcan phosphorylate tau at the pathological sites. Because phosphorylationreleases tau from microtubules and because tau in the PHF is highlyphosphorylated, kinases have been viewed suspiciously for a possiblerole in pathogenesis. There is increasing evidence that CDK5 and GSK3ámay be involved in the pathogenesis of several neurodegenerativedisorders. In neurons that no longer divide, deregulation of Cdks,especially Cdk5, occurs in many neurological disorders, includingAlzheimer's disease (AD) and Parkinson's disease (PD). Fath et al. hasshown that replacement of certain amino acids at known sites ofphosphorylation with a charged amino acid created‘pseudohyper-phosphorylated’ tau that can mimic structural andfunctional aspects of hyperphosphorylated tau (35). In vivo evidence foran interaction with tau exists for Cdk5 and Gsk3. Over-expression ofhuman p25 (an activator of Cdk5) in mice induced tauhyperphosphorylation and cytoskeletal disruptions reminiscent of AD, butno filamentous deposits (36). Noble et al. crossed transgenic miceover-expressing the Cdk5 activator p25, with transgenic miceover-expressing mutant (P301L) human tau. Tau was hyperphosphorylated atseveral sites in the double transgenics, and a highly significantaccumulation of aggregated tau occurred in the brainstem and cortex.Increased numbers of silver-stained neurofibrillary tangles (NFTs)accompanied these changes as well as an association of active GSK withinsoluble tau (37). Over-expression of GSK-3 under the control of atetracycline sensitive transactivator also induced tauhyperphosphorylation, somatodendritic mislocalization of tau, andneuronal apoptosis (38). Recent studies have shown that the β-amyloidpeptide (AP) induces a deregulation of Cdk5 in cultured brain cells, andraises the question on the possible roles of this tauphosphorylatingprotein kinase in the sequence of molecular events leading to neuronaldeath triggered by Aβ. In this context, there is evidence that Cdk5 isinvolved in tau hyperphosphorylation promoted by Aβ in its oligomericform (42). Cdk5 inhibitors protect hippocampal neurons against both tauanomalous phosphorylations and neuronal death. The links between thestudies on the Cdk5/p35 system in normal neurogenesis and its claimedparticipation in neurodegeneration, provide the framework to understandthe regulatory relevance of this kinase system, and changes in itsregulation that may be implicated in disturbances such as thoseoccurring in Alzheimer disease (70). Overall these studies implicate tauhyper-phosphorylation in tau-related neurodegeneration and allude toCdk5, Gsk3 and MAPK as major players in the process.

As demonstrated in the examples set forth below, small molecule purinescaffold compounds are able to inactive the kinases involved in tauphosphorylation and when the appropriate substitution patterns areselected are able to cross the blood brain barrier. Further, addition ofPU24FCl Hsp90 inhibitor to a panel of transformed cells led to adose-dependent induction of Hsp70 and Hsp40. This phenomenon occurred inall the tested cell lines irrespective of their tissue of provenance andwas duplicated in rat cortical primary neurons. Doses of PU24FCl andPU29FCl (another early PU-class compound) that induce a stress responsewere not toxic against normal cells, as demonstrated in a panel ofnormal epithelial and fibroblast cells.

Application of the Invention to Other Neurodegenerative Diseases

Amyotrophic lateral sclerosis is a neurological disorder thatselectively affects motor neurons of brain and spinal cord. Amyotrophiclateral sclerosis (ALS) is characterized by a progressive degenerationof motor neurons that results in severe weakness and skeletal muscleatrophy. The disease is progressive and patients usually succumb tobulbar paralysis, cachexia or respiratory failure within 2-5 years ofonset (44). A distinguishing feature of ALS is the accumulation ofneurofilaments in the perikarya and axons of the spinal motor neurons(for review see Julien 2001, 45). NF—H and NF-M are substrates of CDK5,and the motor neuron inclusion bodies that occur in ALS cases containhyperphosphorylated NF—H (for review see Julien 1999, 47). Emergingevidence indicates an involvement of the serine/threoninecyclin-dependent kinase 5 (Cdk5) in the pathogenesis. Deregulation ofCdk5 by its truncated coactivators, p25 and p29, contributes toneurodegeneration by altering the phosphorylation state of cytosolic andcytoskeletal proteins and, possibly, through the induction of cell cycleregulators.

Parkinson's disease is characterized by bradykinesia in most patientsand many patients may develop a resting tremor (for review see Fahn2003, 48). Classic pathological findings include loss of neuromelanincontaining neurons within the substantia nigra and the presence Lewybodies (48). The Lewy body is an cosinophilic cytoplasmic neuronalinclusion (for review see Fahn 2003, 48), and CDK5 immunoreactivityoccurs in Lewy bodies in the midbrain of Parkinson's disease patients(22). In rats, induction of apoptosis in neurons of the substantia nigraresulted in increased CDK5 levels and activity at the later stages ofapoptosis (49). Further, CDK5 and p35 immunoreactivity was observed inthe perikaryon and nuclei of apoptotic neurons, whereas immunoreactivityin healthy neurons was confined to the axons (49).

Other kinases that are also deregulated in PD, and for which pathogenicmutations have been identified in sporadic PD patients are strongcandidates as HSP90 clients. These include leucine-rich repeat kinase-2(LRRK2) gene were pathogenic mutations cause autosomal-dominant andcertain cases of sporadic Parkinson disease. The G2019S substitution inLRRK2 is the most common genetic determinant of Parkinson diseaseidentified so far, and maps to a specific region of the kinase domaincalled the activation segment. Here we show that autophosphorylation ofLRRK2 is an intermolecular reaction and targets two residues within theactivation segment. The prominent pathogenic G2019S mutation in LRRK2results in altered autophosphorylation, and increasedautophosphorylation and substrate phosphorylation, through a processthat seems to involve reorganization of the activation segment. Anothermutant kinase in the PTEN induced putative kinase 1 (PINK1) gene. Thesemutations were originally discovered in three pedigrees with recessivelyinherited PD. Two homozygous PINK1 mutations were initially identified:a truncating nonsense mutation (W437X) and a G309D missense mutation.Subsequently, multiple additional types of PD-linked mutations ortruncations in PINK1 have been reported, making PINK1 the second mostcommon causative gene of recessive PD. Interestingly, despite autosomalrecessive transmission of PINK-linked early-onset PD, a number ofheterozygous mutations affecting only one PINK allele have beenassociated with late-onset PD. The pathogenic mechanisms by which PINKmutations lead to neurodegeneration are unknown.

PINK1 encodes a 581-amino-acid protein with a predicted N-terminalmitochondrial targeting sequence and a conserved serine/threonine kinasedomain. PINK1 protein has been shown to localize in the mitochondria andexhibit autophosphorylation activity in vitro. The in vivo substrate(s)and biochemical function of PINK1 remain unknown. In cultured mammaliancells, overexpression of wild-type PINK1 protects cells againstapoptotic stimuli whereas small interfering RNA (siRNA)-mediateddepletion of PINK1 increases the susceptibility to apoptotic cell death.In Drosophila, loss of PINK1 leads to mitochondrial defects anddegeneration of muscle and dopaminergic neurons. Despite ample evidenceindicating an essential role of PINK1 in cytoprotection, the mechanismby which PINK1 protects against apoptosis is not understood.

Our results showed that at least Cdk5 and P35 are client proteins ofHsp90. Inhibition of Hsp90 could decrease Cdk5/P35 protein level invitro and P35 level in vivo. Since accumulated evidence implicate thatCdk5/P35 is related to those neurodegenerative diseases. Hsp90 inhibitorcan also be used in the treatment of those diseases.

The invention will now be further described with reference to thefollowing, non-limiting examples.

Example 1

Juvenile mice: Four- to six-week old nu/nu athymic female mice wereobtained from the National Cancer Institute-Frederick Cancer Center andmaintained in ventilated caging. Experiments were carried out under anInstitutional Animal Care and Use Committee-approved protocol, andinstitutional guidelines for the proper and humane use of animals inresearch were followed. Before administration, a solution of PU24FCl wasprepared at desired concentration in 50 μL vehicle (PBS:DMSO:EtOH at1:1:1 ratio). In experiments designed to define the short-term effectsof PU24FCl on tau phosphorylation, mice (2 per time point) were treatedwith 200 mg/kg PU24FCl or with vehicle alone. At the time of sacrifice,brains were collected and immediately flash frozen. For protein analysisbrains were homogenized in SDS lysis buffer (50 mM Tris pH 7.4, 2% SDS).For long-term administration studies, mice (n=5) were treated everyother day for 30 days with the indicated doses of PU24FCl. Weight andbehavior changes were monitored for all animals. Mice were sacrificed byCO₂ euthanasia at 8 h post-last PU24FCl injection. Brains were collectedand processed as mentioned above. Proteins were further analyzed byWestern blot.

Phosphorylation of tau in juvenile and embryonic brains is enhanced (50)and similar to AD afflicted brain (51; 52). Further, nude athymic mice4-6 weeks of age may express tau phosphorylated at relevant diseaseepitopes In a first in vivo experiment, the short term modulation ofHsp90 in the brains of these animals was evaluated. One dose of PU24FCl(200 mg/kg) was administered intraperitoneally to these mice and animalswere sacrificed at 0, 6, 12, 24, 36 and 48 hours. Whole brains werehomogenized in lysing buffer and tau phosphorylation at S202/T205 wasevaluated by Western blot. A burst in tau phosphorylation at thisepitope was observed 12 h post-administration, with a decline to basallevels shortly after (FIG. 1A). Drug levels in the brain tissue wereanalyzed by LC-MS and showed the presence in brain tissue attherapeutically relevant levels with a spike at around 24 hours (FIG.1B). In these same mice, PU24FCl was quickly cleared from the liver,serum and uterus.

In a second experiment, we analyzed the effects of long-term Hsp90inhibition on tau phosphorylation Mice were treated on alternate daysfor 30 days with PU24FCl without observing remarkable toxicity or weightloss in these animals. As seen in FIG. 2, a significant decrease in tauphosphorylation at S202/T205 was evident in all treated mice. Suchdifference in effects between short and long term modulation of Hsp90has been documented for other proteins chaperoned by Hsp90. Treatment ofcells with Hsp90 inhibitors caused degradation of Raf-1 over a long timecourse, while inducing a transient burst of Raf-1 activity whenadministered for a short time (53). Similar evidence has beendemonstrated for the activity of the RNAdependent kinase PKR, whichbecomes active upon short treatment with GM (54). These observationssuggest that Hsp90 may act to restrain the basal signaling of thesekinases. Additional examples are found in the regulation of steroidhormone receptors. Hsp90 masks dimerization and inhibits DNA binding ofsteroid hormone receptors until chaperone interactions are interrupted,typically as a consequence of hormone binding. Thus, steroid hormonereceptors stripped from chaperones are competent for dimerization andDNA binding in the absence of hormone (55). While this function of Hsp90may not hold true for all its client proteins, in the case of p35/cdk5,Hsp90 may undertake a similar role restraining the intrinsic activity ofthe complex, while retaining it in a primed conformation, ready forinteraction with tau.

Reduction in tau phosphorylation in the long-term treatment experimentwas associated with a 60 to 70% decrease in p35 expression (FIG. 2). Inaddition, an increase in the expression of the inducible Hsp70 wasobserved in these mice (FIG. 2). Expression of cdk5 in the whole brainwas not affected. The cdk5 protein is widely distributed in mammaliantissues and in cultured cell lines and is complexed with an array ofother proteins, with each association serving a diverse cellular role.The cdk5/p35 associated kinase activity has been demonstrated only inthe cerebral cortex (56, 57). When immunoprecipitated cdk5 activity wasexamined in AD brains it was found to be elevated in the prefrontalcortex (58). The limited localization of p35/cdk5 in the cortex mayexplain why total cdk5 expression in the whole brain was unchanged uponHsp90 inhibition. Very likely, the high background caused by cdk5localized to other compartments made impossible monitoring a smallchange in cdk5 steady-states by Western blot. These results may alsosuggest that management of cdk5 by Hsp90 in the brain is likely limitedto regulating the activity of the p35/cdk5 complex.

Example 2

Transgenic mice: Transgenic mice, JNPL3 line (59) overexpressing mutanthuman tau (P301L, 4RON) were used in this study. Mice were heterozygousand on a mixed hybrid genetic background composed of C57BL/DBA2/SW, aspublished in ref. 59. These mice develop NFTs in the basaltelencephalon, diencephalon, brainstem, and spinal cord, with severepathology accompanied by degeneration in the spinal cord leading todystonia, paralysis, and death in mice >12 months in age. Nine month-oldmale JNPL3 mice (n=2) were treated intraperitoneally with PU-DZ8 orvehicle for 5 days. Mice were sacrificed 12 h after last treatment bycervical dislocation under anesthesia.

To further examine the effect of Hsp90 on tau phosphorylation, we usedthe JNPL3 line of mice expressing mutant (P301L) tau protein (59).Genetic analyses have linked mutations in the tau gene to FTDP-17 (60,61). Over 20 distinct pathogenic mutations have been identified, withP301L as the most common mutation in tauophaties (33). JNPL3 miceexhibit an age and gene-dose-dependent increase in tau phosphorylationand development of NFTs (59, 62). The tau protein in JNPL3 ispredominantly human and is phosphorylated at multiple sites: T181(AT270), S202/T205 (AT8), T212 (AT100), T231 (AT180), S262, S396/S404,S409 and S422 (59, 62). In concordance with the experiments in thejuvenile nude athymic mice, a five day treatment of nine-month old maleJNPL3 mice with PU-DZ8, a water soluble PU24FCl derivative (2), reducedp35 levels in whole brains and led to a significant amelioration of tauphosphorylation at the putative cdk5 sites, S202/T205 and T212. Thedegree of p35 expression translated well into alleviation ofphosphorylation. A 50% reduction in p35 levels translated inapproximately similar effect on S202/T205 (Ab AT-8), while reducingphosphorylation on T212/S214 (Ab AT-100) almost completely. Nosignificant effect on tau phosphorylated at T231 (Ab AT-180), associatedwith tau in PHF and tangles (63, 64) was seen at a reduction by 50% inp35 expression. However, in mice where effects were more prominent andp35 expression declined to approximately 20% as compared to control, asignificant effect on tau phosphorylation at S202/T205 and T212/S214 anda 50% reduction on T231 was observed. We could not detect a significantamount of tau phosphorylation at T181, site found to behyperphosphorylated in PHF, tangles and neurofilaments (65). Again,whole brain expression of cdk5 was not affected (FIG. 3).

Pharmacologically relevant levels of PU-DZ8 were recorded in thesebrains.

Example 3

JNPL3 female mice 6.5 months of age were treated for 30 days, 5day/week, with the Hsp90 inhibitor PU-DZ8 (FIG. 5) or vehicle, orsacrificed for time zero, n=4/group. Brains were divided in subcorticaland cortical regions and processed using the Greenberg and Daviesextraction protocol. (77) Sarkosyl soluble fractions (S1) were analyzedby WB for p35 and Hsp70, and for tau epitopes found abnormallyhyperphosphorylated in AD brains such as: S202 and T205 recognized byAT8, T181 by AT270, T231 by AT180. These are putative cdk5/p35 sites.Protein bands were normalized to Hsp90 and plotted as relative units.The results are shown in FIGS. 8A and B. Since tauopathy, characterizedby pathogenic phosphorylation of tau can be due to aberrant kinaseactivity, the hsp90 inhibitor is effective because it affects theexpression of the p35 protein, an activator of cdk5 known tophosphorylate tau at pathogenic sites, and thus alleviates tauphosphorylation at these sites.

Example 4

JNPL3 female mice 6 months of age were treated IP with the Hsp90inhibitor PU-DZ8 (75 mg/kg) and sacrificed various times as indicated inFIG. 9. Brains were divided in subcortical and cortical regions andprocessed using the Greenberg and Davies extraction protocol (77).Sarkosyl soluble fractions (S1) extracted from the subcortical regionwere analyzed by WB for p35, cdk5, mutant tau (HT7), Hsp90 and Hsp70.Protein bands were normalized to actin and plotted as relative changefrom untreated mice. FIG. 9 shows degradation of the mutant protein,mtau (HT7) after one dose administration of DZ8. It also shows thechange in chaperone levels (hsp70 increase) and kinase expression (p35levels).

Example 5

COS-7 cells were transfected with cDNAs corresponding to WT and mTau andcells were further treated with PU24FCl for 24 h. Cells were lysed andprotein content analyzed by Western blot. The results are shown in FIG.10. As shown, the mutant Tau (P301L) is very sensitive to the Hsp90inhibitor PU24FCl, while the WT tau is unaffected by similar doses ofdrug.

Example 6

The ability of composition according to the inventions Hsp90 inhibitorsto bind Hsp90 was tested using a fluorescence polarization assaydeveloped by Chiosis et al (WO2005012482, 66, 67, 68). SK—N—SHneuroblastoma cells were treated with Hsp90 inhibitors for 24 h andHsp70 levels were detected by a phenotypic cell-based assay developed byChiosis et al (WO2005012482,69). The results are summarized in FIGS. 11Aand B. As shown, the inhibitors induce a stress response in the SK—N—SHneuroblastoma cells and Hsp70 induction by Hsp90 inhibitors correlateswith their potency in binding to the ATP-regulatory pocket of the Hsp90chaperone.

Example 7

Embryonic primary rat cortical neurons and COS-7 cells transfected withcDNAs corresponding to either p35 alone (COS-7/p35) or both p35 and Tau(COS-7/p35/Tau) are relevant experimental systems to study aberrantneuronal kinase activity because phosphorylation of Tau at putative cdk5sites is both enhanced in these cells and in embryonic and juvenilebrains (50, 52) and is similar to that in AD-afflicted brains (50).COS-7 cells transfected with cDNAs corresponding to either humanWT Tau(COS-7/Tau) or Tau harboring the P301L mutation characteristic offrontotemporal dementia and parkinsonism linked to chromosome 7(COS-7/TauP301L) are cellular models that may be used to differentiatethe effect of Hsp90 inhibition on a mutant protein compared with itsnormal counterpart.

To further examine the roles played by Hsp90 in tauopathy, we made useof both PU24FCl and 17-(allyllamino)-17-demethoxygeldanamycin (17AAG)and investigated their effects on both cdk5/p35 and TauP301L in primaryneuronal and COS-7 cell cultures. Primary neuronal cultures were derivedfrom the cerebral cortices of embryonic day 17 rat embryos andmaintained as described previously (105). To determine the effects ofPU24FCl on protein steady-states and on Tau phosphorylation, PU24FCl wasadded at day 6 of culture, and cells were incubated at 37° C. asindicated. COS-7 cells grown in DMEM with 10% FBS andpenicillin/streptomycin (50 units and 50 μg/ml, respectively) weretransiently transfected by using FuGENE 6 reagent (Roche MolecularBiochemicals, Indianapolis, Ind.) to overexpress p35 and either WT Tauor Tau harboring a P301L mutation. At 12 h after transfection, cellswere incubated for 24 h with the indicated concentration of PU24FCl.After incubation, cells were harvested and lysed in 2%, SDS, and theresulting samples were analyzed by Western blotting.

Phosphorylation of Tau by cdk5 is initiated through activation bycomplex formation with one of the neuron-specific proteins p35 or p39.However, only suppression of p35 by antisense oligonucleotide treatmentbut not of the highly related isoform p39 selectively reduces cdk5activity. In addition, levels of p35 but not of cdk5 protein arerate-limiting for cdk5 activity. In concordance, we assessed theinfluence of Hsp90 inhibition on p35 cellular expression. A dose- andtime-dependent degradation of p35 by PU24FCl was detected in primaryneurons by immunoblot and by immunofluorescence techniques, as well asin COS-7/p35 and COS-7/p35/Tau cells. Effects were seen at ˜1-5 μMPU24FCl and were maximal at 10 μM Hsp90 inhibitor, in agreement with theaffinity of this compound for Hsp90. Exogenously introduced p35 was moresensitive to Hsp90 inhibition than the endogenous protein, suggestingthat by analogy to Hsp90 oncoproteins, buffering and stabilization ofaberrant proteins in tauopathy may be accomplished by co-opting Hsp90.Reduction of p35 levels by Hsp90 inhibition affected the activity of thecdk5/p35 complex, as measured by using a substrate of cdk5, thehistone-H1, and lessened Tau phosphorylation at putative cdk5 shown tobe phosphorylated in AD brains without affecting normal Tau proteinexpression, mTau however, was sensitive to concentrations of PU24FClthat did not interfere with WT Tau expression. The higher sensitivity toHsp90 inhibition of mTau compared with WT Tau is in agreement with theobserved lability of the mutant oncoprotein clients of Hsp90. Analogouseffects on p35 and mTau were observed with 17AAG. The effect of PU24FClon neuronal proteins was well-defined and selective, as the expressionof several kinases and phosphatases that regulate normal Tau activity(PKA, CK-1, CK-2, PP-1-alpha. PP-1-gamma, and PP2A) was not affected bythe Hsp90 inhibitor.

Induction of Hsp70 by Hsp90 inhibitors is documented in severalneurodegenerative disease models (12, 16, 19). Expression of Hsp70 isindirectly regulated by Hsp90 (7). Accordingly, treatment of eitherprimary neurons or transfected COS-7 cells with PU24FCl led to adose-dependent increase in Hsp70. Induction of Hsp70 occurred at dosesof PU24FCl that also modulated both p35 and mTau, suggesting thatdegradation of aberrant proteins and induction of a heat-shock responseare both direct consequences of Hsp90 inhibition by PU24FCl.

Example 8

To examine whether Hsp90 plays a direct role in maintaining thestability of these p35 and mTau, we tested whether inhibition of Hsp90function by PU24FCl affected their half-life. Primary neuronal cultureswere treated with inhibitor or vehicle in the presence of cycloheximide.Quantification of protein levels demonstrated that the half-life ofendogenous p35 was 120 min in the presence of vehicle and decreased to60 min when PU24FCl was added to the system. The exogenous p35 was morelabile and had a significantly shorter half-life than the endogenousprotein (t_(1/2)=60 min in the presence of vehicle and 30 min in thepresence of PU24FCl) for both COS-7/p35/Tau cells and primary neurons.Similar results were observed for mTau: whereas 50% of the protein wasdegraded at 2-4 h in the presence of the Hsp90 inhibitor, the half-lifeof mTau in vehicle treated cells exceeded 10 h. The inhibitor had noeffect on the level of WT Tau. Moreover, mTau and p35 were degraded uponPU24FCl treatment even when induction of Hsp70 was blocked bycycloheximide. These findings strongly position Hsp90 as a direct andimportant regulator of both p35 and mutant Tau stability.

Example 9

To examine whether Hsp90 regulates the stability of these proteinsthrough protein complex formation, we made use of several chemical andimmunological tools that selectively bind either Hsp90 or its putativeclient proteins. Association of Hsp90 with p35 as well as with mTau, wasobserved. No significant association was observed when cells wereimmunopurified with a control IgG. Cdc37, a cochaperone of Hsp90 foundassociated with several chaperone-kinase assemblies, was absent in thep35-immunopurified complexes, in concordance with previous observationsof Lamphere et al. (106). Pretreatment of cells with PU24FCl altered theinteraction of Hsp90 with p35.

The cellular models presented above demonstrate that an interactionbetween Hsp90 and aberrant neuronal proteins is possible at a molecularlevel. However, exogenous introduction of proteins by transfection, maydestabilize the cell's protein content and impose a regulation of thealien protein's stability by Hsp90. Therefore, to evaluate theinteraction of Hsp90 with TauP301L and p35 in an endogenous environment,we made use of brain homogenates obtained from animal models oftauopathy. The JNPL3 line of mice expressing mutant (P301L) human Tau(hTau) protein exhibit an age, gender and gene dose-dependent increasein Tau phosphorylation and insoluble Tau deposits. To isolate proteinsassociated with Hsp90 in these brains, we made use of brain homogenatesobtained from female JNPL3 mice (n=4) 10 months of age and used either abiotinylated PU derivative immobilized on streptavidin beads or aspecific anti-Hsp90 antibody. Hsp90 isolated by PU beads bound mTauspecifically. The presence of the C terminus of heat-shock cognate70-interacting protein, an ubiquitin E3 ligase found to collaborate withmolecular chaperones in facilitating protein folding, was alsoidentified in the Hsp90 complex, in agreement with findings of Sahara etal. (62). An Hsp90 antibody specifically identified the chaperone incomplex with p35 and its kinase partner cdk5. Collectively, these dataposition Hsp90 as a regulator of p35 and mTau stability through directprotein complex formation.

Example 10

Binding to JNPL3 brain Hsp90. The assay buffer (HFB) contained 20 mMHEPES (K) pH 7.3, 50 mM KCl, 5 mM MgCl₂, 20 mM Na₂MoO₄, 0.01% NP40.Before each use, 0.1 mg/mL bovine gamma globulin (BGG) (PanveraCorporation, Madison, Wis.) and 2 mM DTT (Fisher Biotech, Fair Lawn,N.J.) were freshly added. GM-cy3B, a specific Hsp90 ligand, wassynthesized as previously reported (10) and was dissolved in DMSO toform 10 μM solutions. Brains were homogenized in HFB with added proteaseand phosphatase inhibitors. Saturation curves were recorded in whichGM-cy3B (3 nM) was treated with increasing amounts of brain homogenates.The Hill and Scatchard plot analyses of the experiment were constructedto show that at the low amounts of brain homogenates required to reachsaturation, interaction from other cellular material was precluded. Theamount of brain homogenate for which over 90% of GM-cy3B was Hsp90 boundat equilibrium (24 h) was chosen for the competition study. For thecompetition experiments, each 96-well contained 3 nM GM-cy3B, brainhomogenate and tested inhibitor (initial stock in DMSO) in a finalvolume of 100 μL. The plate was left on a shaker at 4° C. for 24 h andthe fluorescence polarization values in mP were recorded. EC₅₀ valueswere determined as the competitor concentrations at which 50% of GM-cy3Bwas displaced. Fluorescence polarization measurements were performed onan Analyst GT instrument (Molecular Devices, Sunnyvale, Calif.). ForGM-cy3B, an excitation filter at 545 nm and an emission filter at 610 to675 nm were used with a dichroic mirror of 565 nm. Measurements weretaken in black 96-well microtiter plates.

FIG. 12 shows the binding affinity of PU-DZ8, PU24FCl and 17AAG to hsp90in JNPL3 brain extracts determined using this procedure. As shown, theEC₅₀ for PU-DZ8 is lower than that of the other compounds. (46.71 nM, asopposed to 822.6 nM for PU24FCl and 98.40 nM for 17AAG).

The same procedure was repeated using the compounds of FIG. 5. The EC₅₀values determined for these compounds are set forth in Table 1. Hsp70induction in neuroblastoma cells by the various purine scaffoldcompounds was determined. The potency for hsp70 induction corresponds tothe hsp90 binding affinity.

TABLE 1 EC50 JNPL3 brain Hsp90 binding Compound (nM) PU-H71 (multiplemeasurements) 15.2, 30.8 PU-DZ8 (multiple measurements) 85.3, 40.1PU-HZ150 5.7 PU-HZ151 6.9 PU-DZ13 8.1 PU-DZ14 8.4 PU-HT65 212.9 PU-HT64122.4 PU-DZ10 192.8 PU-HT70 146.6 PU-HT78 9561 PU-HT133 812.9 PU-BSI1239.3 PU-BSI8 30.2 PU-BSI6 60.8 PU-BSI11 29.9 PU-BSI7 43.5 PU-BSI13 44.5PU-BSI14 42.8 PU-BSI5 26.8 PU-BSI10 105.4 PU-BSI3 199.4 PU-BSI15 122.2PU-BSI16 202.4 PU-BSI4 155.2 PU-DZ12 219.0 PU-DZ16 35.6 PU-DZ15 165.6PU-DZ17 92.3 PU-DZ18 107.1

Example 11

Assessment of PU-DZ8 brain levels. Concentrations of compound weredetermined and quantitated by a MRM mode using a tandem high-performanceliquid chromatography-mass/mass spectrometry (HPLC/MS/MS). A weighedpiece of brain was rinsed with saline isotonic solution, dried withgauze and then homogenized in mobile phase (acetonitrile (ACN)/0.1formic acid=1.2/2.8, v/v). Haloperidol was added as internal standard.PU-DZ8 was extracted in methylene chloride, the organic layer wasseparated, speedily dried under vacuum and reconstituted in the mobilephase. Compound analysis was performed in the API 4000™ LC/MS/MS(Applied Biosystems) which was coupled with a Shimadzu LC system and a96-well plate autosampler. A Gemini C18 column (5μ particle size, 50×4.6mm I.D.) was used for the LC separation. The analyte was eluted under anisocratic condition for 4 min at a flow rate of 0.4 mL/min.

One dose of PU-DZ8 (75 mg/kg) was administered intraperitoncally (i.p.)to female mice of 2.5-4 months in age (n=32) and animals were sacrificedin the interval of 0 to 36 h. Both aggregate-free Tau (S1) and insolubleTau (P3) fractions were prepared from the subcortical and corticalregions of these mice. PU-DZ8 levels in the brain reached 0.35 μg/g(˜700 nM) at 4 h, and the pharmacologically relevant dose was retainedfor at least 12 h post-administration (0.2 μg/g, ˜390 nM). The resultsare shown in FIG. 13.

FIG. 13 shows that PU-DZ8 reaches pharmacologically relevantconcentrations in JNPL3 transgenic mouse brain following administrationof one dose of 75 mg/kg PU-DZ8 administered i.p. This shows that PU-DZ8arrives in the brain tissue much sooner than PU24FCl (FIG. 1B).

Example 12

In a cluster of tauopathies termed “frontotemporal dementia andparkinsonism linked to chromosome 17 (FTDP-17)”, transformation iscaused by several mutations in human Tau isoforms on chromosome 17, thatresult in and are characterized by the accumulation of aggregated Tausimilar to that in AD (10, 11). Over 20 distinct pathogenic mutationshave been identified, with P301L as the most common mutation intauopathies.

To investigate whether release of mTau and p35 from Hsp90 regulationrestores normal neuronal activity and results in elimination of toxicTau aggregates, we made use of the JNPL3 mouse model of tauopathy. Braintissues of JNPL3 mice contain Tau proteins with different solubilitiesand these can be separated into buffer-extractable (S1), high-saltextractable (S2) and sarkosyl-insoluble (P3) fractions. The S1 fractionscontain a 50-60 kDa human Tau protein, whereas sarkosyl-insoluble Tauproteins of 64 kDa and higher molecular weights are detected in thesubcortical brain regions of JNPL3 mice as curly as 3 months inhemizygous females. These contain insoluble toxic Tau phosphorylated atmultiple sites such as T181, S202/T205, T212 and T231 (37, 38).

To investigate whether the human TauP301L present in the JNPL3 line ofmice is a sensitive target for Hsp90 inhibition, animals were treatedwith the brain-barrier permeable Hsp90 inhibitor PU-DZ8. This agent is ahigher-potency water soluble derivative of PU24FCl (EC50JNPL3 brainHsp90=70 nM). One dose of PU-DZ8 (75 mg/kg) was administeredintraperitoneally (i.p.) to female mice of 2.5-4 months in age (n=32)and animals were sacrificed in the interval of 0 to 36 h. Bothaggregate-free Tau (S1) and insoluble Tau (P3) fractions were preparedfrom the subcortical and cortical regions of these mice. Human Taulevels were assessed by immunobloting with a human specific anti-Tauantibody (HT-7). At 4 h post-administration, the Hsp90 inhibitor induceda significant decrease in the soluble precursor pool mTau as present inthe subcortical brain regions (P=0.0031 at 4 h), with these effectsmaintained up to 36 h (P=0.0066 at 8 h, 0.0030 at 12 h, 0.0111 at 24 hand 011.042 at 36 h) (FIG. 14A). We next examined in a 4- to 6-month oldmouse group (n=15) whether the stability of mTau as present in tauaggregates (P3 fraction) was additionally regulated by Hsp90. Asdemonstrated in FIG. 14B, a significant reduction of insoluble(P<0.0001) and hyperphosphorylated (P=0.001) Tau was observed in treatedmice (n=8), as compared to those mice receiving no Hsp90 inhibitor(n=7).

No significant changes in cdk5 expression were detected, indicating thatmanagement of cdk5 by Hsp90 in the brain may be limited to regulatingthe activity of the p35/cdk5 complex. The expressions of Akt and Raf-1,nodal proteins in cell survival and growth pathways, respectively,tightly regulated by Hsp90 in malignant cells were not altered byPU-DZ8.

For experiments designed to test the kinetics of mTau and p35 modulationby Hsp90 inhibitors, animals were administered intraperitoneally (i.p.)75 mg/kg PU-DZ8 in PBS (6% DMSO). Mice were sacrificed by CO2 euthanasiaat the indicated times following PU-DZ8 administration. Hemibrains wereseparated into cortico-limbic (cortex, amygdale and hippocampus) andsubcortical (basal ganglia, diencephalon, brain stem and cerebellum)regions, quickly frozen on dry ice and stored at −80° C. and processed.In summary, each brain piece was weighed and homogenized in threevolumes of Tris-buffered saline (TBS) containing protease andphosphatase inhibitors (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA,1 mM EGTA, 5 mM sodium pyrophosphate, 30 mM 3-glycerophosphate, 30 mMsodium fluoride, 1 mM phenylmethylsulfonyl fluoride (PMSF)). Thehomogenates were centrifuged at 27,000 g for 15 min at 4° C.Supernatants were collected as S1 fractions, and the pellets (P1) werere-homogenized in three volumes of salt/sucrose buffer (0.8 M NaCl, 10%sucrose, 10 mM Tris/HCl, pH 7.4, 1 mM EGTA, 1 mM PMSF) and centrifugedas above. The resulting pellets were discarded and the supernatants wereincubated with sarkosyl (Sigma, St Louis. Mo. USA; 1% finalconcentration) for 1 h at 37° C. The sarkosyl mixtures were thencentrifuged at 150,000 g for 30 min at 4° C. The supernatants (S2fraction) were collected, and the pellets (P3) were resuspended in 50E4L 2% SDS in TBS and stored at −80° C. for western blotting. Proteinswere analyzed by Western blot.

FIG. 14A, shows the effects of one dose, short term administration ofPU-DZ8 on the levels of soluble mutant tau in the JNPL3 mouse brain Thesubcortical brain region of 2.5 to 4-month old mice is presented. HumanTau levels were normalized to those of Hsp90. FIG. 14B shows the effectof one dose, short-term administration of PU-DZ8 on the levels ofinsoluble mutant tau in the JNPL3 mouse brain. Analysis of the insolubletau (P3) fractions extracted from the subcortical brain region of6-month old mice treated with PU-DZ8 (75 mg/kg) for 4, 8, 12 and 24 h ispresented

Example 13

To investigate whether modulation of mTau could be sustained over alonger Hsp90 inhibitor-treatment period, without being toxic to mice.JNPL3 mice were subjected for 30 days to these agents. Female JNPL3 mice6.5 months of age (n=10) were administered i.p. vehicle (n=5) or one ofthe Hsp90 inhibitors. PU24FCl (200 mg/kg) or PU-DZ8 (75 mg/kg) (n=5), ona daily, five-times per week schedule and animals were sacrificed at 8 hfollowing the last administered dose of inhibitor. No toxicity wasobserved as evidenced by lack of significant change in animal weight,fur appearance, appetite and posture. Furthermore, no visible internalorgan damage was detected at sacrifice upon gross inspection. Both S1and P3 fractions extracted from the subcortical brain region of thesemice were analyzed for mTau expression and phosphorylation. Asignificant reduction in Tau expression and phosphorylation in both theprecursor protein pool (S1 fraction) (hTau, P<0.0001) and the toxicaggregate (P3 fraction) (phosphorylated Tau at T231, P=0.0034) (FIG.15), as well as p35 reduction in S1 fraction was observed in micetreated with the Hsp90 inhibitor.

Collectively, the rapid kinetics of Tau degradation in both the solublepool and the aggregated form by the Hsp90 inhibitors suggests that Hsp90regulates the toxic Tau aggregate and facilitates its formation andaccumulation. These data also suggest that an Hsp90 inhibitor may beused in the treatment of tauopathies both to prevent the formation oftoxic aggregates and to solubilize the already aggregated toxic tau.

FIG. 15 shows the effect of long term PU-DZ8 administration onhyperphosphorylated tau in toxic tau aggregates.

Example 14

In tauopathies transformation is characterized by abnormalities in theTau protein leading to an accumulation of hyperphosphorylated andaggregated Tau (5-7). In Alzheimer's disease (AD), Tauhyperphosphorylation is suggested to be a pathogenic process caused byaberrant activation of several kinases, in particular cyclin-dependentprotein kinase 5 (cdk5) and glycogen synthase kinase-3 beta (gsk3β,leading to phosphorylation of Tau on pathogenic sites.Hyperphosphorylated Tau in AD is believed to misfold, undergo netdissociation from microtubules and form toxic Tau aggregates (9, 10).Phosphorylation of Tau by cdk5 is initiated through activation bycomplex formation with one of the neuron-specific proteins p35 or p39(22, 23). However, only suppression of p35 by antisense oligonucleotidetreatment, and not of the highly related isoform p39, selectivelyreduces cdk5 activity (24). In addition, levels of p35 but not cdk5protein are rate-limiting for cdk5 activity (25). In concordance, weassessed the influence of Hsp90 inhibition on p35 expression.

We detected a dose- and time-dependent degradation of p35 by PU24FCl inprimary neurons, as well as in COS-7/p35 and COS-7/p35/Tau cells.Embryonic primary rat conical neurons and COS-7 cells transfected withcDNAs corresponding to either p35 alone (COS-7/p35) or both p35 and Tau(COS-7/p35/Tau) are cellular systems that enable the evaluation of theseinhibitors on cdk5/p35 activity and stability and also on Tauphosphorylation at putative cdk5 sites. These are relevant experimentalsystems to study aberrant neuronal kinase activity becausephosphorylation of Tau at these sites is enhanced in embryonic andjuvenile brains (20) and is similar to AD afflicted brains (21). Inaddition. COS-7 cells transfected with the cdk5 activator p35 expressTau phosphorylated at pathogenic sites (21). Effects were seen atapproximately 1-5 μM PU24FCl and were maximal at 10 LM Hsp90 inhibitor,in agreement with the affinity of this compound for Hsp90. Exogenouslyintroduced p35 was more sensitive to Hsp90 inhibition than theendogenous protein, suggesting that by analogy to Hsp90 oncoproteins,buffering and stabilization of aberrant proteins in tauopathy may beaccomplished by co-opting Hsp90. Reduction of p35 levels by Hsp90inhibition affected the activity of the cdk5/p35 complex, as measuredusing a substrate of cdk5, the histone-H1.

To investigate whether decreased p35 expression resulted in reducedphosphorylation of Tau, we measured Tau phosphorylation on threeputative cdk5 sites, namely S202/T205, T231 and T181 (26, 27). Thesesites have been shown to be abnormally phosphorylated in AD brains (28).PU24FCl lessened phosphorylation on these sites in a dose-dependentmanner without affecting normal Tau protein expression. As observed forp35 levels and activity, effects were evident at 5 μM and maximal at 10μM inhibitor. In addition, the kinetics of p35 degradation were similarto those observed for reduction in Tau phosphorylation.

To investigate the in vivo effect of Hsp90 inhibition on p35 in a WT Tauenvironment, we made use of hTau mice (41). hTau mice develop Taupathology with a distribution that is comparable to that occurring inthe early stages of AD. The majority of Tau pathology in hTau mice islocated in the cortical brain region. These mice express six isoforms ofnon-mutant human Tau, but develop AD-like Tau-pathology. Heat-stablefractions (S1) prepared from cortical homogenates of these mice indicatean age-related accumulation of Tau phosphorylated at putative cdk5sites. We examined whether inhibition of Hsp90 in these brains may leadto a reduction in p35 expression and a consequent alleviation of Tauphosphorylation. hTau female mice (n=10) 4 and 8-10 months of age wereadministered either vehicle or one dose of PU-DZ8 (75 mg/kg) i.p. andanimals were sacrificed at 4 h or 8 h post-administration.Aggregate-free Tau (S1) fractions were prepared from the cortical regionof these mice and human Tau levels assessed by immunobloting with anantibody specific for 3-repeat domain Tau (RD3). By analogy toexperiments on primary neuronal cultures and WT Tau transfected cells,the Hsp90 inhibitor had no effect on soluble WT Tau expression. However,both a significant time-dependent reduction in p35 levels (P=0.0019)(FIG. 16A) and alleviation of Tau phosphorylation on Ser202, as detectedby antibody CP13 (P=0.0078), were evident at 8 h post-administration ofthe Hsp90 inhibitor (FIG. 16B). The monoclonal antibody CP13 is commonlyused to detect Tau pathology in both early and more advanced stages ofTau aggregate accumulation (41). Collectively, these data positionp35/cdk5 as a kinase complex prone to aberrantly phosphorylate WT andmutant Tau, and suggest Hsp90 as a regulator of its activity in both Tauenvironments.

FIG. 16A shows the effect of PU-DZ8 on p35 in the htau mice that expresspathogenically hyperphosphorylated WT tau similarly to Alzheimer'spatients. FIG. 16B shows the effect of PU-DZ8 tau phosphorylation in thehtau mice that express pathogenically hyperphosphorylated WT tausimilarly to Alzheimer's patients.

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The following references are cited herein, and are incorporated hereinby reference in their entirety.

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1. A method for treatment of neurodegenerative disease, comprising thestep of administering to an individual in need of such treatment atherapeutically effective amount of a purine scaffold compound thatinhibits Hsp90, wherein the compound and the mode of administration areselected such that the compound is delivered to the brain.
 2. The methodof claim 1, wherein the compound crosses the blood brain barrier.
 3. Themethod of claim 1 or 2, wherein the purine scaffold compound is acompound comprising a purine moiety to which is bonded an additionalaryl or heteroaryl ring at the 8- or 9-position via a linker, whereinthe compound as a whole possesses the necessary flexibility andsubstituent groups to be received within the N-terminal pocket of Hsp90.4. The method of claim 3, wherein the additional aryl or heteroaryl ringis affixed at the 9-position and is substituted at the 4′ and 5′positions only.
 5. The method of claim 3, in which the purine scaffoldcompound has the general structure:

wherein R is hydrogen, a C₁ to C₁₀ alkyl, alkenyl, alkynyl, or analkoxyalkyl group, optionally including heteroatoms such as N or O; Y₁and Y₂ are independently C, N, S or O, with the proviso that when Y₁and/or Y₂ is O the double bonds are missing or rearranged to retain thearyl nature of the ring X₄ is hydrogen, halogen, for example F or Cl, orBr; X₃ is CH₂, CF₂, S, SO, SO₂, O, NH, or NR², wherein R² is alkyl; andX₂ is halogen, alkyl, halogenated alkyl, alkoxy, halogenated alkoxy,hydroxyalkyl, pyrollyl, optionally substituted aryloxy, alkylamino,dialylamino, carbamyl, amido, alkylamido dialkylamido, acylamino,alkylsulfonylamido, trihalomethoxy, trihalocarbon, thioalkyl, SO₂,alkyl, COO-alkyl, NH₂, OH₂, or CN; and X₁ represents one moresubstituents on the aryl group, with the proviso that X₁ represents atleast one substituent in the 5′-position said substituent in the5′-position being selected from the same choices as X₂: C₁ to C₆ alkylor alkoxy; or wherein X₁ has the formula —X—Y—Z— wherein X, Y and Z areindependently C, N, S or O, connected by single or double bonds and withappropriate hydrogen substitution to satisfy valence, and Y may be(CH₂)₂, and one of X and Z is bonded at the 5′-position of the aryl ringand the other is bonded to the 4′ position.
 6. The method of claim 5,wherein the right-side aryl group is substituted at the 2′ and 5′position only.
 7. The method of claim 5, wherein the right side arylgroup is substituted at the 2′, 4′, and 5′ positions.
 8. The method ofclaim 5, wherein at least one of X, Y and Z is a carbon atom.
 9. Themethod of claim 8, wherein X₁ is —O—(CH₂)_(n)—O—, wherein n is 1 or 2.10. The method of claim 9, wherein X₂ is halogen.
 11. The method ofclaim 10, wherein X₂ is Br or I.
 12. The method of claim 9, wherein R isan alkyl group containing a nitrogen heteroatom.
 13. The method of claim12, wherein R is 3-isopropylaminopropyl,3-(isopropyl(methyl)amino)propyl, 3-(isopropyl(ethyl)amino)propyl,3-((2-hydroxyethyl)(isopropyl)amino)propyl,3-(methyl(prop-2-ynyl)amino)propyl, 3-(allyl(methyl)amino)propyl,3-(ethyl(methyl)amino)propyl, 3-(cyclopropyl(propyl)amino)propyl,3-(cyclohexyl(2-hydroxyethyl)amino)propyl,3-(2-methylaziridin-1-yl)propyl, 3-(piperidin-1-yl)propyl,3-(4-(2-hydroxyethyl)piperazin-1-yl)propyl, 3-morpholinopropyl,3-(trimethylammonio)propyl, 2-(isopropylamino)ethyl,2-(isobutylamino)ethyl, 2-(neopentylamino)ethyl,2-(cyclopropylmethylamino)ethyl, 2-(ethyl(methyl)amino)ethyl,2-(isobutyl(methyl)amino)ethyl, or 2-(methyl(prop-2-ynyl)amino)ethyl.14. The method of claim 13, wherein R is 3-isopropylaminopropyl.
 15. Themethod of claim 13, wherein X₂ is halogen.
 16. The method of claim 15,wherein X₂ is Br or I.
 17. The method of claim 9, wherein X₄ is halogen.18. The method of claim 17, wherein X₂ is halogen.
 19. The method ofclaim 18, wherein X₂ is Br or I.
 20. The method of claim 17, wherein Ris an alkyl group containing a nitrogen heteroatom.
 21. The method ofclaim 20, wherein R is 3-isopropylaminopropyl,3-(isopropyl(methyl)amino)propyl, 3-(isopropyl(ethyl)amino)propyl,3-((2-hydroxyethyl)(isopropyl)amino)propyl,3-(methyl(prop-2-ynyl)amino)propyl, 3-(allyl(methyl)amino)propyl,3-(ethyl(methyl)amino)propyl, 3-(cyclopropyl(propyl)amino)propyl,3-(cyclohexyl(2-hydroxyethyl)amino)propyl,3-(2-methylaziridin-1-yl)propyl, 3-(piperidin-1-yl)propyl,3-(4-(2-hydroxyethyl)piperazin-1-yl)propyl, 3-morpholinopropyl,3-(trimethylammonio)propyl, 2-(isopropylamino)ethyl,2-(isobutylamino)ethyl, 2-(neopentylamino)ethyl,2-(cyclopropylmethylamino)ethyl, 2-(ethyl(methyl)amino)ethyl,2-(isobutyl(methyl)amino)ethyl, or 2-(methyl(prop-2-ynyl)amino)ethyl.22. The method of claim 21, wherein R is 3-isopropylaminopropyl.
 23. Themethod of claim 21, wherein X₂ is halogen.
 24. The method of claim 23,wherein X₂ is Br or I.
 25. The method of claim 9, wherein R is aterminal alkyne.
 26. The method of claim 25, wherein R is propynyl. 27.The method of claim 26, wherein X₂ is halogen.
 28. The method of claim1, wherein the purine scaffold compound has the formula:


29. The method of claim 1 or 2, wherein the purine scaffold compound hasthe formula:

wherein R is an alkyl, alkenyl, alkynyl, or an alkoxyalkyl group,optionally including heteroatoms such as N or O connected to the 2′position to form an 8 or 10 membered ring ring; Y₁ and Y₂ areindependently C, N, S or O, with the proviso that when Y₁ and/or Y₂ is Othe double bonds are missing or rearranged to retain the aryl nature ofthe ring X₄ is hydrogen, halogen, for example F or Cl, or Br; X₃ is CH₂,CF₂, S, SO, SO₂, O, NH, or NR², wherein R² is alkyl; and X₂ is part ofR; and X₁ represents one more substituents on the aryl group, with theproviso that X₁ represents at least one substituent in the 5′-positionsaid substituent in the 5′-position being selected from the same choicesas X₂: C₁ to C₆ alkyl or alkoxy; or wherein X₁ has the formula —X—Y—Z—wherein X, Y and Z are independently C, N, S or O, connected by singleor double bonds and with appropriate hydrogen substitution to satisfyvalence, and Y may be (CH₂)₂, and one of X and Z is bonded at the5′-position of the aryl ring and the other is bonded to the 4′ position.30. The method of claim 1, wherein the purine scaffold compound has theformula